Inhibition of H1N1 by Picochlorum sp. 122 via AKT and p53 signaling pathways

Abstract Influenza viruses cause a severe threat to global health, which can lead to annual epidemics and cause pandemics occasionally. However, the number of anti‐influenza therapeutic agents is very limited. Polysaccharides, extracted from Picochlorum sp. (PPE), seaweed Polysaccharides, have exhibited antiviral activity and were expected to be used for influenza treatment. In our research, the capability of PPE to inhibit H1N1 infection was proved in MDCK cells. PPE could make MDCK cells avoid being infected with H1N1 and inhibited nuclear fragmentation and condensation of chromatin. PPE evidently inhibited the generation of reactive oxygen species in MDCK cells. Mechanism study revealed that PPE prevented MDCK cells from H1N1 infection through induction of apoptosis by stimulating AKT signaling pathway and suppressing p‐p53 signaling pathway. In conclusion, PPE turns out to act as a prospective antiviral drug for H1N1 influenza.

in an attempt to control these influenza, while the availability of influenza vaccines shows little effect against the infection (Feng et al., 2014;Hu et al., 2022;Shang et al., 2021). There are many important compounds that can be synthesized by Algal products, polysaccharide (algae-based polymer), lipid, and organic pigment, for example. These have attracted kinds of attention in industrial applications (Schutter et al., 2010). Besides, Algal is used as food supplements, aquaculture ingredients, animal feed, and soil biofertilizers (Li et al., 2013;Lian et al., 2018;Nie et al., 2016). Seaweed polysaccharide can be served as many important materials used in clinical practice, including anti-inflammatory activity, anticancer activity, wound dressings, drug delivery, tissue engineering, immunomodulators, antibacterial effects, and anticoagulant activity, which are attributed to their rich sources, high safety, great biological activity, and low side effects (de Jesus Raposo et al., 2015;Kholiya et al., 2020;Okimura et al., 2020). Polysaccharides have shown the capability to inhibit viral infection in some studies, and they mainly inhibit virus from attaching cell wall to make cells avoid being invaded by virus and inhibit virus proliferation (Abu-Galiyun et al., 2019;Liang et al., 2021;Sharma et al., 2021). The source of PPT as previously described (Guo et al., 2021;Xie et al., 2017;Xu et al., 2021;Yao et al., 2022). However, there are few reports about the antiviral activity. The mechanism of antiviral drugs against H1N1 infection has attracted researchers' attention increasingly. Li reported that functionalized selenium nanoparticles decorated with amantadine were reported to inhibit H1N1-induced apoptosis via the ROS-mediated AKT signaling pathway . Wang found that Selenium Nanoparticles decorated with β-Thujaplicin could inhibit apoptosis induced by H1N1 influenza virus via ROS-mediated p53 and AKT Signaling Pathways . Mou discovered that EGCG induces β-defensin 3 against H1N1 by the MAPK signaling pathway (Ha et al., 2022;Mou et al., 2020;Zhu et al., 2022). The anti-H1N1 function of PPE was demonstrated, and the apoptosis mechanism referring to ROS-mediated signaling pathways was discovered in the research. Our research showed that PPE inhibited host cell apoptosis induced by H1N1 through the AKT, P53, and PARP signaling pathways.

| Cell viability
MTT assay was used to test the cytotoxicity of PPE as previously described . MDCK cells were inoculated at 5 × 10 4 / well and then cultured for 24 h. After that, H1N1 viruses were added with titer of 100 TCID 50 for 2 h and removed which out of the cells.

TA B L E 1 Titer of H1N1
The cells were treated with H1N1 for 24 h before rinsed twice with PBS, and each well were added with 15 μl of MTT (5 mg/ml) solution.
After 4 h, the formed formazan crystals were dissolved by dimethyl sulfoxide (DMSO) at 150 μl/well and detected at 570 nm.

| Annexin-V/PI double staining assay
MDCK cells were cultured and harvested as described previously . Briefly, the collected cells were suspended and then centrifuged. The supernatant solution was removed and the cells were suspended again. Finally, the cells were stained with the kits for 10-20 min without light and tested by flow cytometry, and Cell Quest VTM software was used to analyze the data.

| The assessment of reactive oxygen species (ROS)
ROS in MDCK cells were detected as previously described . In brief, after H1N1-infected MDCK cells were exposed to PPE for 24 h, MDCK cells were stained by the kits, and the results were tested with fluorescence microscope and fluorescence plate reader.

| Test of mitochondrial membrane potential (△Ψm)
JC-1 monomers was used to detect mitochondrial membrane potential (△Ψm) . H1N1-infected MDCK cells were stained with JC-1 without light for 20 min after exposed to PPE for 24 h. Flow cytometry was utilized.

| TUNEL and DAPI staining
Effects of PPE on DNA fragmentation induced by H1N1 were detected by the kits following the protocol. Briefly, MDCK cells were labeled by TUNEL and nuclear was stained by DAPI. Then, the stained cells were observed with a fluorescence microscope (Nikon Eclipse 80i).

| Statistical analysis
All the data are presented as mean ± SD. Differences between the two groups were evaluated using two-tailed Student's t-test. Oneway analysis of variance was used in multiple-group comparisons.

| In vitro antiviral effect of PPE
Cell viability was tested to assess PPE on antiviral effects by MTT assay. In Figure 1a, PPE exhibited low cytotoxicity against MDCK cells. In Figure 1b

| Effects of PPE on mitochondrial function of H1N1-infected MDCK cells
Reactive oxygen species (ROS) are proved as a significant regulator of cell apoptosis induced by chemotherapy drugs. Mitochondrion acts as an important organelle to produce intracellular ROS.
Overproduction of ROS can reduce ATP synthesized by mitochondria and result in mitochondrial dysfunction which leads to cell apoptosis further (Li et al., 2011). As shown in Figure 2a, H1N1

| Effects of PPE on apoptosis in H1N1-infected MDCK cells
In order to explore whether apoptosis was induced in H1N1-infected MDCK cells and effects of PPE, further steps were taken. As shown in Figure 3a, there is an obvious sub-G1 peak ( concentrations showed a greater capability to inhibit apoptosis.
Significant differences were not observed in cell cycle distribution.
DNA fragmentation is one of the representatives of cell apoptosis (Moosavi et al., 2018). The apoptosis induced by H1N1 was further confirmed by TUNEL and DAPI assay. As shown in Figure 3b

| Effects of PPE on the early and late apoptosis of H1N1-infected MDCK cells
Annexin-V/PI double staining acts as a more effective way for cell apoptosis examination (Yu et al., 2020). As shown in Figure 4a, H1N1 infection resulted in a significant elevation of green and red fluorescence of MDCK cells, which indicated that H1N1 induced early and late apoptosis of H1N1-infected MDCK cells. After treatment with PPE, the double fluorescence showed a decrease obviously.
The results indicated that PPE suppressed viral activity in a dosedependent manner, and exhibited great antiviral capability to prevent MDCK cells from undergoing apoptosis in vitro.

| The innate immune response modulation of PPE to H1N1 infection
In addition, PPE exerted an anti-inflammatory activity. In Figure 5, MDCK cells showed a basal secretion of cytokines. H1N1 infection induced a strong innate immune response in the hosts such as TNFα, IL-2, IL-17F, IL-4, IL-17A, and IL-1β, while hosts treated with PPE showed significant suppression of innate immune response.

| Effects of PPE on apoptotic signaling pathways activated by ROS
The overexpression of ROS could cause damage to DNA via regulating apoptosis signaling pathways. In Figure 6a,  AKT, and P-P53 signaling pathways (Figure 6b).

| CON CLUS IONS
In our research, the antiviral activity and mechanisms of PPE were explored by different methods. The research demonstrated that PPE obviously restrained H1N1 proliferation and reduced apoptosis of MDCK cells. The mechanisms revealed that PPE limited H1N1induced apoptosis of hosts through AKT and p53 signaling pathways, and suppressed the innate immune response of MDCK cells induced by H1N1. Our findings revealed that PPE could be a prospective drug to treat H1N1 infection. Medica (LMM2020-7).

CO N FLI C T O F I NTE R E S T
The authors report no conflicts of interest in this work.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data sharing is not applicable to this article as no new data were created or analyzed in this study.